While antibody-based therapeutics have emerged as a major component in clinical applications, the generation of antibodies to important targets such as cell surface receptors, ion channels, and glycoproteins remains difficult. These proteins contain buried functional sites that are unreachable by conventional mouse or human IgG-based antibodies. Single domain antibodies have shown a promising ability to target difficult antigens and hidden epitopes. Dozens of single domain antibodies are currently being evaluated in various stages of clinical trials. Dr. Mitchell Ho at the NCI has demonstrated that single domain antibodies are capable of targeting buried functional sites in cancer signaling complexes [Feng et al. PNAS, 2013; Gao et al Nature Communications, 2015; Li et al. PNAS, 2017]. The Ho lab has constructed large shark and camel single-domain ('nanobody') libraries and isolated binders to a wide range of antigens [Feng et al. Antibody Therapeutics, 2019], indicating that the phage-displayed single domain antibody libraries can be a valuable source to isolate therapeutic antibodies. In FY19, Dr. Ho's lab reported the construction of a large phage-displayed VNAR single-domain antibody library from six nurse sharks in the journal Antibody Therapeutics [Feng et al. 2019; PMID: 30627698; doi: 10.1093/abt/tby011]. Shark new antigen receptor variable domain (VNAR) antibodies can bind restricted epitopes that may be inaccessible to conventional antibodies. The Ho lab developed a library construction method based on polymerase chain reaction (PCR)-Extension Assembly and Self-Ligation (named EASeL) to construct a large VNAR antibody library with a size of 1010 from six adult nurse sharks (Ginglymostoma cirratum) in collaboration with Dr. Martin Flajnik at the University of Maryland Baltimore. The next-generation sequencing analysis of 1 million full-length VNARs revealed that this library is highly diversified because it covers all four classical VNAR types (Types I-IV) including 11% of classical Type I and 57% of classical Type II. About 30% of the total VNARs could not be categorized as any of the classical types. The high variability of complementarity determining region (CDR) 3 length and cysteine numbers are important for the diversity of VNARs. To validate the use of the shark VNAR library for antibody discovery, we isolated a panel of VNAR phage binders to cancer therapy-related antigens, including glypican-3, human epidermal growth factor receptor 2 (HER2), and programmed cell death-1 (PD1). Additionally, we identified binders to viral antigens that included the Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) spike proteins. The isolated shark single-domain antibodies including Type I and Type II VNARs were produced in Escherichia coli and validated for their antigen binding. A Type II VNAR (B6) has a high affinity (Kd = 10 nM) for its antigen. Our work demonstrates that the nurse shark VNAR library is a useful source for isolating single-domain antibodies to a wide range of antigens. The EASeL method may be applicable to the construction of other large diversity gene expression libraries. In FY19, Dr. Ho has been invited to speak at multiple international meetings (e.g. PEGS, PepTalk, Antibody Engineering & Therapeutics, SCBA, CBA) about his work on single domain antibodies and their applications in cancer immunotherapy [Zhang et al. Antibody Therapeutics, 2018; PMID: 30406214; doi: 10.1093/abt/tby009]. Although antibody engineering technologies may require further optimization, the methodology is well established and, in many cases, validated clinically. Dr. Ho suggested that one area of research that may lead to a significant paradigm change in antibody engineering should be the development of single-domain antibodies ('nanobodies') such as camelid VHH and shark VNAR [Ho Antibody Therapeutics, 2018; PMID: 30101214; doi: 10.1093/abt/tby001]. These antibodies represent a new and very different class of products: small, easy to express and produce, stable, and capable of penetrating tissues to reach buried sites in tumor or viral antigens. In recent years, the Ho lab has developed a group of human single-domain antibodies that target tumor-specific antigens such as mesothelin, GPC2, and GPC3 with high affinity. The single-domain antibodies have been used to inhibit Wnt, Yap, and other key cancer signaling pathways that are important for cell proliferation. It would be interesting to see whether the new wave of single-domain proteins could eventually become a game changer in our toolbox of magic bullets for treating cancer and other diseases.